WO2012129399A2 - Compositions for and methods of evaluating tumors having a skeletal muscle origin - Google Patents

Abstract

Myoblastic differentiation biomarkers are provided for evaluating tumors having a skeletal muscle origin. In addition, kits are provided for evaluating expression levels or presence of the biomarkers associated with such tumors. Furthermore, methods are provided for evaluating such tumors via measuring expression levels or presence of the biomarkers in the cytoplasm of cells.

Description

COMPOSITIONS FOR AND METHODS OF EVALUATING TUMORS HAVING A

SKELETAL MUSCLE ORIGIN

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of US Provisional Patent Application No. 61/466,289 filed March 22, 201 1 , incorporated herein by reference as if set forth in its entirety.

FIELD OF THE DISCLOSURE

[002] The invention relates generally to the field of medical diagnostics, and more particularly to compositions for and methods of evaluating tumors having a skeletal muscle origin.

BACKGROUND

[003] Rhabdomyosarcoma ("RMS"), in which cancer cells arise from skeletal muscle, is the most common soft tissue sarcoma in children and young adults. Identification of rhabdomyoblasts in tumors of muscle origin or those with rhabdomyoblastic differentiation is pertinent to precise evaluation of these tumors. Though certain morphologic features characterize rhabdomyoblasts, accurate recognition on the basis of cross-striations by light microscopy, or actin and myosin filaments and Z bands by electron microscopy are tedious and often unrewarding. Consequently, an incorrect classification of a tumor by a clinician can lead to a course of therapy that is not appropriate or effective for an individual.

[004] Cells undergoing differentiation to skeletal muscle (i.e., myoblastic differentiation) are indicative of RMS. Current methods of evaluating RMS involve histological examination of biopsied tissue by a pathologist based on morphological characteristics. Immunohistochemical staining for biomarkers indicative of skeletal muscle differentiation, such as desmin, myogenin, MyoD1 and myoglobin, also are utilized to determine if cancer cells are undergoing differentiation to skeletal muscle.

[005] Presently, myogenin and MyoD1 , which are myogenic transcriptional regulatory proteins expressed early in skeletal muscle differentiation, are the most sensitive and specific markers for RMS. However, myogenin also can be detected in rare, non-RMS tumors and normal skeletal muscle. As such, methods of detecting myogenin alone do not provide a clinician with a definitive diagnostic tool for RMS. Likewise, methods of detecting MyoDI alone do not provide the clinician with a definitive diagnostic tool for RMS.

[006] In view of the foregoing, a need exists in the art for additional compositions and methods for evaluating tumors having a skeletal muscle origin such as RMS.

BRIEF SUMMARY

[007] Compositions are provided for detecting a tissue undergoing myoblastic differentiation or for diagnosing, differentiating, prognosing or monitoring a tumor having a skeletal muscle origin. In one aspect, a kit is provided for detecting cytoplasmic expression of at least one myoblastic differentiation biomarker. In another aspect, a kit is provided for diagnosing, differentiating, prognosing or monitoring a tumor having a skeletal muscle origin that expresses at least one myoblastic differentiation biomarker.

[008] The kits can include a means for processing a biological sample, a means for isolating biomarker nucleic acid or amino acid molecules, and a means for detecting nucleic acid or amino acid molecule biomarker levels {e.g., isolation buffers, colorimetric assay buffers and reagents, and other detection buffers and reagents).

[009] The kits also can include positive and/or negative controls for the at least one myoblastic differentiation biomarker. For example, the kits can include at least a means for measuring myoblastic differentiation biomarkers such as p63, myogenin, MyoDI, desmin or myoglobin. Alternatively, the kits can include at least a means for measuring p63 and at least one of myogenin, MyoDI, desmin or myoglobin. Alternatively, the kits can include at least a means for measuring p63.

[0010] Methods are provided for detecting myoblastic differentiation or for diagnosing, differentiating, prognosing or monitoring a tumor having a skeletal muscle origin. In one aspect, a method is provided for detecting myoblastic differentiation. The method can include measuring cytoplasmic expression of at least one myoblastic differentiation biomarker in cells from a biological sample, where cytoplasmic expression of the at least one biomarker in the cells indicates that the cells are undergoing myoblastic differentiation.

[0011] In another aspect, a method is provided for diagnosing an individual as having a tumor having a skeletal muscle origin. The method can include detecting cytoplasmic expression of at least one myoblastic differentiation biomarker in cells from a biological sample from the individual, where cytoplasmic expression of the at least one biomarker in the cells indicates that the individual has the tumor.

[0012] In another aspect, a method is provided for differentiating a tumor having a skeletal muscle origin from a tumor not having a skeletal muscle origin. The method can include detecting cytoplasmic expression of at least one myoblastic differentiation biomarker in cells from a biological sample, where cytoplasmic expression of the at least one biomarker in the cells indicates that the tumor has a skeletal muscle origin, or where lack of cytoplasmic expression of the at least one biomarker indicates that the tumor does not have a skeletal muscle origin.

[0013] In another aspect, a method is provided for prognosing an individual having or suspected of having a tumor having a skeletal muscle origin. The method can include detecting cytoplasmic expression of at least one myoblastic differentiation biomarker in cells from a biological sample from the individual, where elevated cytoplasmic expression of the at least one biomarker in the cells when compared to a control/reference indicates a poor prognosis, or where attenuated cytoplasmic expression of the at least one biomarker in the cells when compared to a control/reference indicates a good/favorable prognosis.

[0014] In another aspect, a method is provided for monitoring progression of a tumor having a skeletal muscle origin in an individual having the tumor. The method can include detecting cytoplasmic expression of at least one myoblastic differentiation biomarker in cells from first and second chronological biological samples from the individual, where elevated cytoplasmic expression of the at least one biomarker in cells of the second chronological sample when compared to cytoplasmic expression of the at least one biomarker in cells of the first chronological sample indicates tumor progression, or where attenuated cytoplasmic expression of the at least one biomarker in cells of the second chronological sample when compared to cytoplasmic expression of the at least one biomarker in cells of the first chronological sample indicates tumor regression.

[0015] In another aspect, a method is provided for monitoring effectiveness of a therapy in an individual having a tumor having a skeletal muscle origin. The method can include detecting cytoplasmic expression of at least one myoblastic differentiation biomarker in cells from at least two chronological biological samples from the individual, where elevated cytoplasmic expression of the at least one biomarker in cells in a later- obtained chronological sample when compared to cytoplasmic expression of the at least one biomarker in an earlier-obtained sample indicates an ineffectiveness of therapy, or where attenuated cytoplasmic expression of the at least one biomarker in cells in a later-obtained chronological sample when compared to cytoplasmic expression of the at least one biomarker in an earlier-obtained sample indicates an effectiveness of therapy. At least one of the later-obtained biological samples can be obtained after an appropriate therapy has been administered to the individual.

[0016] In the methods, the at least one myoblastic differentiation biomarker expression can be determined at a nucleic acid or amino acid level. The at least one myoblastic differentiation biomarker can be selected from p63, desmin, myogenin, MyoD1 and myoglobin; p63, desmin, myogenin and MyoD1 ; p63, desmin, myogenin and myoglobin; p63, desmin, MyoD1 and myoglobin; p63, myogenin, MyoD1 and myoglobin; p63, desmin and myogenin; p63, desmin and MyoD1 ; p63, desmin and myoglobin; p63, myogenin and MyoD1 ; p63, myogenin and myoglobin; p63, MyoD1 and myoglobin; p63 and desmin; p63 and myogenin; p63 and MyoD1 ; p63 and myoglobin; and p63 only. As such, at least one of the myoblastic differentiation biomarkers is p63.

[0017] The methods also can include obtaining a biological sample from an individual having or suspected of having a tumor having a skeletal muscle origin.

[0018] The methods also can include processing the biological sample from the individual.

[0019] The methods also can include morphologically evaluating the cells such as under a microscope.

[0020] The methods also can include selecting an appropriate therapy or adjusting an existing therapy in an individual. [0021] The compositions and methods therefore find use in detecting, diagnosing, differentiating, prognosing and monitoring tumors having a skeletal muscle origin, which can be used to select an appropriate therapy or to follow the effectiveness of such a therapy.

[0022] These and other features, objects and advantages of the present invention will become better understood from the description that follows. In the description, reference is made to the accompanying drawings, which form a part hereof and in which there is shown by way of illustration, not limitation, embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The features, objects and advantages other than those set forth above will become more readily apparent when consideration is given to the description below. Such description makes reference to the following drawings, wherein:

[0024] FIG. 1 shows p63 immunostaining demonstrates well-defined cross-striations in RMS (a and b, 800 x), medullomyoblastoma (c, 800 x), rhabdomyomatous Wilms tumor (d, 800 x), rhabdomyoma (e, 800 x), and cardiac muscle (f, 800 x).

[0025] FIG. 2 shows comparative p63 and desmin immunohistochemistry in RMS (a, d and g, H&E, 800 x). p63 immunostain shows distinct cross-striations (c, f and i, 800 x), whereas desmin does not (b, e and h, 800 x).

[0026] FIG. 3 shows immunoelectron microscopy of p63 localizing to the Z band of the sarcomere.

[0027] FIG. 4 shows a comparison of spindle cell RMS (a-c, H&E, 800 x) and leiomyosarcoma (d-f, H&E, 800 x). Both entities show immunoreactivity for desmin (b and e, 800 x), but p63-stained cross-striations are visible only in the spindle cell RMS (c, 800 x). Cross-striations are not seen in the leiomyosarcoma stained with p63 (f, 800 x).

[0028] While the invention is susceptible to various modifications and alternative forms, exemplary embodiments thereof are shown by way of example in the drawings and are described in detail below. It should be understood, however, that the description of exemplary embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the invention as defined by the embodiments herein and appended claims. Reference therefore should be made to the embodiments herein and appended claims for interpreting the scope of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0029] The compositions and methods now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments are shown.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which the invention pertains. Although any materials and methods similar to or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are described herein.

[0031] Moreover, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that there be one and only one element. The indefinite article "a" or "an" thus usually means "at least one."

[0032] Overview

[0033] One challenge in medicine is providing accurate and reliable methods of identifying a disease or distinguishing diseases from one another. The work described herein is the first to show intense cytoplasmic staining of the myoblastic differentiation biomarker, p63, in tissue undergoing myoblastic differentiation. The work therefore provides a basis for compositions and methods for detecting, diagnosing, differentiating, prognosing and monitoring a tumor having a skeletal muscle origin. Advantageously, the compositions and methods described herein are useful in a variety of applications including, but not limited to, identifying myoblastic differentiation in a biological sample, diagnosing a biological sample from an individual having or suspected of having a tumor having a skeletal muscle origin, differentiating tumors having a skeletal muscle origin from tumors having a non-skeletal muscle origin, and prognosing or monitoring tumors having a skeletal muscle origin.

[0034] Tumors Having a Skeletal Muscle Origin

[0035] As used herein, "tumor" means a tissue comprising malignant cells of a cancer. As used herein, "tumor having a skeletal muscle origin" means malignant cells that arise from striated or skeletal muscle cells or progenitor cells thereof. Examples of such a tumor include, but are not limited to, RMS, rhabdomyomas, rhabdomyomatous Wilms tumors, pleuropulmonary blastoma with rhabdomyomatous differentiation, teratoma with atypical rhabdoblasts, medullomyoblastoma, Triton tumor (peripheral nerve sheath tumor with striated muscle differentiation) and any tumor with skeletal muscle differentiation.

[0036] As used herein, "tumor not having a skeletal muscle origin" or "tumor having a non-skeletal muscle origin" means malignant cells that do not arise from striated or skeletal muscle cells, but instead arise from blood cells ("angio" or "hemangio"), bone cells ("osteo"), cartilage cells ("chondro"), fat cells ("lipo"), fibrous tissue cells ("fibro"), nerve cells ("neuro" or "neurofibro"), smooth muscle cells ("leiomyo") or synovial cells. Examples of such a tumor include, but are not limited to, acoustic neuroma, adenocarcinoma, angiosarcoma, astrocytoma, basal cell carcinoma, bile duct carcinoma, bladder carcinoma, breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, colon carcinoma, craniopharyngioma, cystadenocarcinoma, embryonal carcinoma, endotheliosarcoma, ependymoma, epithelial carcinoma, Ewing's tumor, fibrosarcoma, glioma, hemangioblastoma, hepatoma, leiomyomas, leiomyosarcoma, liposarcoma, lung carcinoma, lymphangiosarcoma, lymphangioendotheliosarcoma, medulloblastoma, medullary carcinoma, melanoma, meningioma, mesothelioma, myxosarcoma, neuroblastoma, oligodendroglioma, osteogenic sarcoma, ovarian cancer, papillary adenocarcinomas, papillary carcinoma, pancreatic cancer, prostate cancer, renal cell carcinoma, sebaceous gland carcinoma, seminoma, small cell lung carcinoma, squamous cell carcinoma, sweat gland carcinoma, and germ cell tumors. [0037] TP53 Gene Family and p63

[0038] p63, a member of the TP53 gene family, is essential for the development of limbs, craniofacial structures and several epithelia. Normally, p63 is expressed in the basal epithelial cells of different organs, including the breast, skin, uterine cervix, urogenital tract and prostate. It also been proposed as a possible marker of stem cells or reserve cells. See, Barbareswchi et al. (2001 ) Am. J. Surg. Pathol. 25:1054-1060.

[0039] Nucleic acid and amino acid sequences for p63 are known and characterized. See, e.g., GenBank® Accession Nos. BC039815 (nucleic acid; mRNA) and AAH39815 (amino acid).

[0040] All p53 family members are involved in regulating muscle differentiation through the retinoblastoma ("RB") protein. The early stages of muscle differentiation, up to the expression of myogenin, can occur without the p53 family members. However, later steps involving exit from the cell cycle and activation of muscle-specific gene transcription require an active RB protein. The p53 family members function in separate, but complementary, ways to produce and maintain an active RB protein. For example, p53 is required to induce transcription of the RB gene, whereas p63 and p73 induce the cyclin-dependent kinase inhibitor p57 to maintain RB in an active, hypophosphorylated state. Loss of these p53 family functions by overexpression of dominant negative ("DN") p73 or mutations in TP53 blocks myogenic differentiation, thereby allowing cooperating oncogenes to induce neoplastic transformation in myoblasts. Induction of cellular differentiation, therefore, may offer an explanation for the high frequency of TP53 pathway mutations seen in RMS patients. See, Ikawa et al. (1999) Cell. Death Differ. 6:1 154-1 161 ; and Cam et al. (2006) Cancer Cell 10:281 -293.

[0041] In surgical pathology, antibody to p63, solo or in cocktail combination with other immunohistochemical markers, frequently is used in the diagnosis of prostate carcinoma because normal prostatic glands are lined by p63-positive basal cells, whereas carcinomatous glands lack a basal cell layer. See, Shah et al. (2002) Am. J. Surg. Pathol. 26:1 161 -1 168; Weinstein et al. (2002) Mod. Pathol. 15:1302-1308; and Tacha et al. (2004) Appl. Immunohistochem. Mol. Morph. 12:75-78. p63 immunostaining also is used to support a diagnosis of squamous cell carcinoma in various sites, including the head and neck, lung (Reis-Filho et al. (2003) Virchows Arch . 443:122-132), uterine cervix and anus (Owens et al. (2007) Am. J. Surg. Pathol. 31 :285-290); as well as metaplastic carcinoma of the breast (Koker et al. (2004) Am. J. Surg. Pathol. 28:1506-1512), urothelial carcinoma, sarcomatoid carcinoma and giant cell tumor of bone (Dicksen et al. (2008) Mod. Pathol. 21 :369-375; and Lee et al. (2008) Mod. Pathol. 21 :531 -539). In these applications, however, p63 is localized to the nucleus.

[0042] As disclosed herein, and in contrast to the above applications, elevated cytoplasmic staining of p63 was demonstrated in cells/tissues/tumors undergoing myoblastic (i.e., striated or skeletal muscle) differentiation. The discovery that an elevated cytoplasmic p63 expression pattern in a soft tissue tumor is indicative of tumors such as RMS provides a powerful diagnostic tool for clinicians. Accurate diagnosis of RMS is required to evaluate treatment modalities such as surgical resection, radiotherapy and chemotherapy. The markers that are currently utilized to diagnose RMS are not universally reliable, as underscored by the potential for false positive results that may confound definitive diagnosis. Accordingly, accurate histological classification of tumors has prognostic relevance and can aid in the selection of appropriate therapy.

[0043] Rhabdomyosarcoma

[0044] Of particular interest herein are soft tissue tumors (i.e., sarcomas), particularly RMS, which is composed of malignant cells that arise from a normal skeletal muscle cell. Because skeletal muscle cells are found in virtually every site of the body, RMS can develop in almost any part of the body. RMS therefore is a group of histologically and genetically heterogeneous sarcomas.

[0045] As used herein, "rhabdomyosarcoma" or "RMS" means all types and stages of RMS. Examples of the types of RMS include, but are not limited to, embryonal rhabdomyosarcoma ("ERMS"), alveolar rhabdomyosarcoma ("ARMS"), undifferentiated rhabdomyosarcoma ("URMS"), botryoid rhabdomyosarcoma ("BRMS"), and pleomorphic rhabdomyosarcoma ("PRMS").

[0046] In general, ERMS tends to occur in the head and neck area, bladder, vagina and in or around the prostate and testes, and typically affect infants and young children. ARMS tends to occur more often in large muscles of the trunk, arms and legs, and typically affects older children or teenagers. In ARMS, the malignant cells form little hollow spaces that resemble alveoli. BRMS, a subset of ERMS, tends to occur under mucosal surfaces of body orifices and is commonly observed in areas such as the vagina, bladder and nares. Typically, it is distinguished by the formation of polyploid grapelike tumor masses, and it histologically presents as malignant cells in an abundant myxoid stroma. Lastly, PRMS tends to occur in individuals aged 30-50 years, and it histologically presents as irregularly arranged cells that vary in size, thus its pleomorphic distinction.

[0047] Diagnosing and Staging RMS

[0048] Because RMS is comprised of malignant cells of skeletal muscle origin, diagnosis typically is performed by examining biopsy specimens for morphology and immunohistochemical indicators (i.e., phenotype diagnosis), although genotypic diagnosis also can be performed.

[0049] Once RMS is diagnosed, it can be staged according to Table 1 , although many other staging systems are known in the art. See, e.g., Lawrence et al. (1997) Cancer 80:1 165-1 170; and Pedrick et al. (1986) J. Clin. Oncol. 4:370-378. Staging is the process of determining how far a tumor has spread. Treatment and prognosis therefore depend, to a large extent, on the tumor's stage.

[0050] Table 1 : Site-Modified Tumor, Nodes, Metastasis ("TNM") Staging System

Stage Site T Status Size Node Status Metastasis

1 Favorable T1 or T2 a or b NO, N1 , or NX M0

2 Unfavorable T1 or T2 a NO or NX M0

3 Unfavorable T1 or T2 a N1 M0

3 Unfavorable T1 or T2 b NO, N1 , or NX M0

4 Favorable or Unfavorable T1 or T2 a or b N0 or N1 M1

[0051] Treating RMS

[0052] Treatment options tend to vary depending upon the stage of RMS. Clinicians often use a simpler system based on whether the tumors are likely to be resectable (where all visible tumor can be removed by surgery) or unresectable. Resectability is based on whether the tumor appears to have grown into nearby tissues or spread to distant sites, as well as on whether or not an individual is healthy enough to have surgery.

[0053] Although surgery typically is part of the treatment regimen whenever possible, the treatment of RMS is multi-disciplinary. As such, other forms of treatment include, but are not limited to, chemotherapy, immunotherapy, radiation therapy or a combination thereof (e.g., chemo-radiation therapy).

[0054] Chemotherapy for RMS can be administered via an intravenous line. Typically, chemotherapy lasts 6-12 months, and a chemotherapeutic agent can be administered in about two to about five (or sometimes ten) day "pulses" or "cycles" every 3-4 weeks. Some chemotherapeutic agents can be given on a weekly basis. Examples of chemotherapeutic agents for use in RMS chemotherapy include, but are not limited to, Vincristine, Dactinomycin, Cyclophosphamide, Topotecan, Irinotecan, Etoposide, Ifosfamide, Doxorubicin and Carboplatin.

[0055] Radiation therapy for RMS typically begins after 4-5 cycles of chemotherapy have been given (i.e., after about 12 weeks), although in selected cases radiation therapy may begin at the same time (or as shortly thereafter as possible) as chemotherapy.

[0056] Compositions

[0057] Compositions of the invention can include kits for use in detecting at least one myoblastic differentiation biomarker in a biological sample. As used herein, "kit" means any manufacture {e.g., a package or a container) having at least one means for specifically detecting myoblastic differentiation biomarker expression {e.g., at least one antibody, at least one nucleic acid probe, etc.) and a positive and/or negative control. The kit may be promoted, distributed, or sold as a unit for performing any of the methods of the present invention. The kits therefore can include at least a means for detecting or determining p63 and a positive and/or negative control.

[0058] As used herein, "biomarker" or "biomarkers" means nucleic acid {e.g., gene) or amino acid {e.g., protein) molecules whose level of expression in a cell, tissue, organ or mammal is altered compared to that of a normal or healthy cell, tissue, organ or mammal. The myoblastic differentiation biomarkers described herein have expression levels that correlate with a tumor, particularly a tumor having a skeletal muscle origin such as RMS, and detection, diagnosis, differentiation, prognosis and monitoring thereof.

[0059] The biomarkers can include polynucleotides comprising the entire or partial sequence of the nucleotide sequence encoding the biomarkers, or the complement of such sequences. As used herein, "polynucleotide" means a polymer of nucleic acids or nucleotides that, unless otherwise limited, encompasses naturally occurring bases (i.e., adenine, guanine, cytosine, thymine and uracil) or known base analogues having the essential nature of naturally occurring nucleotides in that they hybridize to single- stranded nucleic acid molecules in a manner similar to naturally occurring nucleotides. Although it may comprise any type of nucleotide units, the term generally applies to nucleic acid polymers of ribonucleic acids ("RNA") or deoxyribonucleic acids ("DNA"). The term includes single-stranded nucleic acid polymers, double-stranded nucleic acid polymers, and RNA and DNA made from nucleotide or nucleoside analogues that can be identified by their nucleic acid sequences, which are generally presented in the 5' to 3' direction (as the coding strand), where the 5' and 3' indicate the linkages formed between the 5' hydroxyl group of one nucleotide and the 3'-hydroxyl group of the next nucleotide. For a coding strand presented in the 5'-3' direction, its complement (or non- coding strand) is the strand that hybridizes to that sequence according to Watson-Crick base pairing. Thus, as used herein, the complement of a nucleic acid is the same as the "reverse complement" and describes the nucleic acid that in its natural form, would be based paired with the nucleic acid in question.

[0060] As used herein, a "nucleic acid," "nucleotide" or "nucleic acid residue" are used interchangeably to mean a nucleic acid that is incorporated into a molecule such as a gene or other polynucleotide. As noted above, the nucleic acid may be a naturally occurring nucleic acid and, unless otherwise limited, may encompass known analogues of natural nucleic acids that can function in a similar manner as naturally occurring nucleic acids. Examples of nucleic acids include any of the known base analogues of DNA and RNA such as, but not limited to, 4-acetylcytosine, 8-hydroxy-N6- methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5- carboxymethylaminonnethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 - methyladenine, 1 -methylpseudouracil, 1 -methylguanine, 1 -methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyanninonnethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5- methyluracil, -uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

[0061] As such, the biomarkers can include DNA or RNA comprising the entire or partial nucleotide sequence thereof.

[0062] Alternatively, the biomarkers can include a peptide, polypeptide or protein encoded by or corresponding to the nucleotide sequence of a biomarker described herein. When the biomarker is a peptide, polypeptide or protein, it can include the entire or partial amino acid sequence of any of the biomarker proteins or polypeptides.

[0063] As used herein, "amino acid" or "amino acid residue" are used interchangeably herein to mean an amino acid that is incorporated into an amino acid molecule such as a peptide, polypeptide or protein (collectively, "protein"). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogues of natural amino acids that can function in a similar manner as naturally occurring amino acids.

[0064] Whether polynucleotides or polypeptides, the biomarkers can include not only the entire biomarker sequence but also fragments and/or variants thereof. As used herein, "fragment" or "fragments" means a portion of the nucleic or amino acid sequence of the biomarker. Polynucleotides that are fragments of a biomarker nucleic acid sequence generally comprise at least about 10, 15, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1 ,000, 1 ,200 or 1 ,500 contiguous nucleotides, or up to the number of nucleotides present in a full-length biomarker polynucleotide disclosed herein. Likewise, a fragment of a biomarker polypeptide comprises at least about 15, 25, 30, 50, 100, 150, 200 or 250 contiguous amino acids, or up to the total number of amino acids present in a full-length biomarker protein.

[0065] As used herein, "about" means within a statistically meaningful range of a value or values such as a stated concentration, length, molecular weight, pH, sequence identity, time frame, temperature or volume. Such a value or range can be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by "about" will depend upon the particular system under study, and can be readily appreciated by one of skill in the art.

[0066] As used herein, "variant" or "variants" means substantially similar sequences. Generally, variants of a particular biomarker have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity (preferably over the full length) to a biomarker as determined by sequence alignment programs.

[0067] One of skill in the art understands that variants can be constructed via modifications to either the polynucleotide or polypeptide sequence of the biomarker and can include substitutions, insertions {e.g., adding no more than ten nucleotides or amino acid) and deletions {e.g., deleting no more than ten nucleotides or amino acids). Methods of mutating and altering nucleic acid sequences, as well as DNA shuffling, are well known in the art. See, e.g., Crameri et al. (1997) Nature Biotech. 15:436-438; Crameri et al. (1998) Nature 391 :288-291 ; Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; Moore et al. (1997) J. Mol. Biol. 272:336-347; Stemmer (1994) Proc. Natl. Acad. Sci. USA 91 :10747-10751 ; Stemmer (1994) Nature 370:389-391 ; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; and Techniques in Molecular Biology (Walker & Gaastra eds., MacMillan Publishing Co. 1983) and the references cited therein; as well as US Patent Nos. 4,873,192; 5,605,793 and 5,837,458.

[0068] Methods of aligning sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers & Miller (1988) CABIOS 4:1 1 -17; the local alignment algorithm of Smith et al. (1981 ) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman & Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local alignment method of Pearson & Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448; the algorithm of Karlin & Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873- 5877.

[0069] As used herein, "means for detecting" or "means for determining" includes using one or more complimentary nucleic acid molecule probes or one or more antibodies specific for the at least one myoblastic differentiation biomarker and means for detecting the binding of the nucleic acid molecule probes or antibodies to the biomarker. As used herein, "probe" means any molecule that is capable of selectively binding to a specifically intended target biomolecule, for example, a nucleotide transcript or a protein encoded by or corresponding to the biomarker. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies and organic molecules.

[0070] Myoblastic differentiation biomarkers for use in the compositions described herein can include, but are not limited to, p63, desmin, myogenin, MyoD1 and myoglobin.

[0071] A nucleic acid molecule for use in the kits can include a polynucleotide sequence (or its complement) that can hybridize to an uninterrupted nucleic acid sequence derived from the biomarker nucleic acid sequence under either stringent or moderately stringent hybridization conditions. Stringent hybridization conditions are commonly defined as hybridizing at 68°C in 5x SSC/5x Denhardt's solution/1 .0% SDS, and washing in 0.2x SSC/0.1 % SDS +/- 100 g/ml denatured salmon sperm DNA at room temperature ("RT"), and moderately stringent hybridization conditions are defined as washing in the same buffer at 42°C. The appropriate stringency for hybridizing nucleic acid molecules depends on the length of the nucleic acids and their degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T_m for hybrids of nucleic acid molecules having those sequences. The relative stability (corresponding to higher T_m) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating T_m have been derived (see, "Molecular Cloning: A Laboratory Manual," 3^rd ed. (Sambrook et al. eds., Cold Spring Harbor Press 2001 )). For hybridization with shorter nucleic acids (i.e., oligonucleotides), the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see, id.). A minimum length for a hybridizable nucleic acid molecule is at least about 10 nucleotides, preferably at least about 15 nucleotides, and more preferably at least about 20 nucleotides. Additional guidance regarding such conditions is readily available in the art in, for example, "Current Protocols in Molecular Biology," (Ausubel et al. eds., John Wiley & Sons 1995).

[0072] Examples of p63 probes can be found in, for example, Gu et al. (2008) Cancer Lett. 263:26-34; Knouf et al. (2012) Nucleic Acids Res. 40:499-510; Laurikkala et al. (2006) Development 133:1553-1563; Lin et al. (2009) PLoS Genet. 5:e1000680; Mills et al. (1999) Nature 398:708-713; Parsa et al. (1999) J. Invest. Derm. 1 13:1099- 1 105; and Parsons et al. (2009) Prostate 69:559-569; see also, US Patent No. 6,972,181 . Likewise, examples of probes for desm in, MyoD1 , myoglobin and myogenin can be found in, for example, Cessna et al. (2001 ) Am. J. Surgical Path. 9:1 150-1 157; Dias et al. (1990) Am. J. Pathol. 137:1283-1291 ; Goldstein et al. (2006) Neoplasia 8:332-343; Kumar et al. (2000) Mod. Pathol. 13:988-993; Scrable et al. (1989) Genes, Chormosomes & Cancer 1 :23-35; Sebire et al. (2003) J. Clin. Pathol. 56:412-416; Wang et al. (1995) Am. J. Pathol. 147:1799-1810; and Wijnaendts et al. (1994) J. Pathol. 174:283-292.

[0073] When making polynucleotides for use as probes to the biomarker (e.g., hybridization probes or primer sets), one of skill in the art can be further guided by knowledge of redundancy in the genetic code as shown below in Table 2. [0074] Table 2: Redundancy in Genetic Code.

Residue Triplet Codons Encoding the Residue

Ala (A) GCU, GCC, GCA, GCG

Arg (R) CGU, CGC, CGA, CGG, AGA, , AGG

Asn (N) AAU, AAC

Asp (D) GAU, GAC

Cys (C) UGU, UGC

Gin (Q) CAA, CAG

Glu (E) GAA, GAG

Gly (G) GGU. , GGC , GGA, GGG

His (H) CAU, CAC

lie (1) AUU, AUC, AUA

Leu (L) UUA, UUG, CUU, CUC, CUA, CUG

Lys (K) AAA, AAG

Met (M) AUG

Phe (F) UUU, UUC

Pro (P) ecu, CCC, CCA, CCG

Ser (S) UCU, UCC, UCA, UCG, AGU, AGC

Thr (T) ACU, ACC, ACA, ACG

Trp (W) UGG

Tyr (Y) UAU, UAC

Val (V) GUU, GUC, GUA, GUG

START AUG

STOP UAG, UGA, UAA

[0075] Methods of synthesizing polynucleotides are well known in the art, such as cloning and digestion of the appropriate sequences, as well as direct chemical synthesis {e.g., ink-jet deposition and electrochemical synthesis). Methods of cloning polynucleotides are described in, for example, Copeland et al. (2001 ) Nat. Rev. Genet. 2:769-779; Current Protocols in Molecular Biology (Ausubel et al. eds., John Wiley & Sons 1995); Molecular Cloning: A Laboratory Manual, 3^rd ed. (Sambrook & Russell eds., Cold Spring Harbor Press 2001 ); and PCR Cloning Protocols, 2^nd ed. (Chen & Janes eds., Humana Press 2002). Methods of direct chemical synthesis of polynucleotides include, but are not limited to, the phosphotriester methods of Reese (1978) Tetrahedron 34:3143-3179 and Narang et al. (1979) Methods Enzymoi. 68:90- 98; the phosphodiester method of Brown et al. (1979) Methods Enzymoi. 68:109-151 ; the diethylphosphoramidate method of Beaucage et al. (1981 ) Tetrahedron Lett. 22:1859-1862; and the solid support methods of Fodor et al. (1991 ) Science 251 :767- 773; Pease et al. (1994) Proc. Natl. Acad. Sci. USA 91 :5022-5026; and Singh-Gasson et al. (1999) Nature Biotechnol. 17:974-978; as well as US Patent No. 4,485,066. See also, Peattie (1979) Proc. Natl. Acad. Sci. USA 76:1760-1764; as well as EP Patent No. 1721908; Int'l Patent Application Publication Nos. WO 2004/022770 and WO 2005/082923; US Patent Application Publication Nos. 2009/0062521 and 201 1/0092685; and US Patent Nos. 6,521 ,427; 6,818,395; 7,521 ,178 and 7,910,726.

[0076] Where the presence of a biomarker protein within a cell is to be detected from, for example, a tissue sample, the means for detecting or determining the biomarker can be an antibody or a functional fragment thereof, as would be used in, for example, flow cytometric analysis, immunochemical detection/localization of the biomarker in tumor cells or other biological samples, and immunoblot analysis {e.g., dot blot, Western blot) of extracts from tumor cells or other biological samples.

[0077] As used herein, "antibody" or "antibodies" includes an immunoglobulin molecule immunologically reactive with a particular antigen, and includes both polyclonal and monoclonal antibodies. The term also includes genetically engineered forms such as chimeric antibodies {e.g., humanized murine antibodies) and heteroconjugate antibodies {e.g., bispecific antibodies). For example, the term includes bivalent or bispecific molecules, diabodies, triabodies and tetrabodies. Bivalent and bispecific molecules are described in, for example, Kostelny et al. (1992) J. Immunol. 148:1547; Pack & Pluckthun (1992) Biochemistry 31 :1579; Zhu et al. (1997) Protein Sci. 6:781 ; Hu et al. (1996) Cancer Res. 56:3055; Adams et al. (1993) Cancer Res. 53:4026; and McCartney et al. (1995) Protein Eng. 8:301 . Antibody also includes antigen binding forms of antibodies, including fragments with antigen-binding capability {e.g., Fab', F(ab')₂, Fab, Fv and rlgG). Treatment of antibodies with proteolytic enzymes, such as papain and pepsin, generates these antibody fragments, especially anti-biomarker fragments. The term also refers to recombinant single chain Fv fragments (scFv). Preferably, antibodies employed to practice the present invention bind to its target protein with an affinity (association constant) of equal to or greater than 10⁷ M^"1.

[0078] Commercially available anti-p63 anti-MyoD1 , anti-myogenin, anti-desmin and anti-myoglobin antibodies can be obtained from, for example, Abeam (Cambridge, MA); BioLegend (San Diego, CA); Chemicon/lnvitrogen (Carlsbad, CA); Dako North America (Carpinteria, CA); Lifespan Biosciences (Seattle, WA); Novus Biologicals (Littleton, CO); Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); Sigma Aldrich (St. Louis, MO); and Spring Biosciences (Pleasanton, CA).

[0079] Alternatively, one of skill in the art also is familiar with methods of making monoclonal or polyclonal antibodies given the availability of a biomarker's amino acid sequence. An antibody can be a monoclonal and polyclonal antibody and can belong to any antibody class (i.e., IgG, IgM, IgA, etc.). For example, one of skill in the art can make monoclonal antibodies by isolating lymphocytes and fusing them with myeloma cells, thereby producing hybridomas. See, e.g., Milstein C, "Handbook of experimental immunology," (Blackwell Scientific Pub., 1986); and Goding J, "Monoclonal antibodies: principles and practice," (Academic Press, 1983). The cloned hybridomas are then screened for production of, for example, "anti-p63" (i.e., antibodies that bind preferentially to p63 or fragments thereof). Monoclonal antibodies are thus not limited by the manner in which the antibodies are produced, whether such production is in situ or not. Alternatively, antibodies can be produced by recombinant DNA technology including, but not limited, to expression in bacteria, yeast, insect cell lines or mammalian cell lines.

[0080] Likewise, one of skill in the art can make polyclonal antibodies by immunizing a suitable host animal, for example, such as a rabbit, with an immunogen or an immunogenic fragment thereof of the biomarker and using properly diluted serum or isolating immunoglobulins from the serum. The animal may therefore be inoculated with the immunogen, with blood subsequently being removed from the animal and an IgG fraction purified. Other suitable host animals include a chicken, goat, sheep, guinea pig, rat or mouse. If desired, the immunogen may be administered as a conjugate in which the immunogen is coupled, for example, via a side chain of one of its amino acid residues, to a suitable carrier. The carrier molecule is typically a physiologically acceptable carrier. The antibody obtained may be purified to a purity of up to about 70%, up to about 80%, up to about 90%, up to about 95%, up to about 99% or up to about 100%.

[0081] When making monoclonal or polyclonal antibodies or functional fragments thereof, the biomarker can be purified or produced recombinantly or by chemical synthesis, and fragments or other derivatives or analogs thereof, including fusion proteins, can be used as an immunogen to generate antibodies that recognize the biomarker. The antibodies can be cross reactive {e.g., they may recognize the biomarker from different species), and polyclonal antibodies have greater likelihood of cross reactivity.

[0082] When making antibodies for use as probes to the biomarker, one of skill in the art can be further guided by knowledge that amino acids within the same conservative group typically can substitute for one another without substantially affecting the function of a peptide, polypeptide or protein (e.g., for use as an antigen or epitope). For the purpose of the present invention, such conservative groups are set forth in Table 3 and are based on shared properties. See also, Alberts et al., "Small molecules, energy, and biosynthesis," 56-57 In: Molecular Biology of the Cell, 3^rd ed. (Garland Publishing Inc. 1994).

[0083] Table 3. Amino Acid Conservative Substitutions.

Residue Side Chain Side Chain pH Hydropathy Preferred

Polarity Index Conservative

Substitution

Ala (A) Non-polar Neutral 1 .8 Ser

Arg (R) Polar Basic (strongly) -4.5 Lys, Gin

Asn (N) Polar Neutral -3.5 Gin, His

Asp (D) Polar Acidic -3.5 Glu

Cys (C) Non-polar Neutral 2.5 Ser

Gin (Q) Polar Neutral -3.5 Asn, Lys

Glu (E) Polar Acidic -3.5 Asp

Gly (G) Non-polar Neutral -0.4 Pro

His (H) Polar Basic (weakly) -3.2 Asn, Gin

lie (I) Non-polar Neutral 4.5 Leu, Val

Leu (L) Non-polar Neutral 3.8 lie, Val

Lys (K) Polar Basic -3.9 Arg, Gin

Met (M) Non-polar Neutral 1 .9 Leu, lie

Phe (F) Non-polar Neutral 2.8 Met, Leu, Tyr

Pro (P) Non-polar Neutral -1 .6 Gly

Ser (S) Polar Neutral -0.8 Thr

Thr (T) Polar Neutral -0.7 Ser

Trp (W) Non-polar Neutral -0.9 Tyr

Tyr (Y) Polar Neutral -1 .3 Trp, Phe

Val (V) Non-polar Neutral 4.2 lie, Leu [0084] The following six groups each contain amino acids that are typical but not necessarily exclusive conservative substitutions for one another: 1 . Alanine (A), Serine (S), Threonine (T); 2. Aspartic acid (D), Glutamic acid (E); 3. Asparagine (N), Glutamine (Q); 4. Arginine (R), Lysine (K); 5. Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6. Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0085] Methods of synthesizing polypeptides or producing them recombinantly are well known in the art. See, e.g., Ausubel et al. (1995), supra; and Proteins: Structures and Molecular Principles (Creighton ed., W. H. Freeman & Co. 1983). Short peptides, for example, can be synthesized on a solid support or in solution. Longer peptides may be made using recombinant DNA techniques. Here, polynucleotides encoding the biomarkers may be synthesized, and/or cloned, and expressed according to methods well known in the art and used in the production of, for example, antibodies for use in the kits described below to detect the biomarkers. See, e.g., Sambrook & Russell (2001 ), supra.

[0086] Substantial changes in function can be made by selecting substitutions that are less conservative than those listed in the table above, i.e., by selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of substitution, (b) the charge or hydrophobicity of the polypeptide at the target site, or (c) the bulk of a side chain. The substitutions that in general can be expected to produce the greatest changes in the polypeptide's properties will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted by a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted by any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl or histidyl, is substituted by an electronegative side chain, e.g., glutamyl or aspartyl; (d) a residue having a bulky side chain, e.g., phyenylalanyl, is substituted by a residue not having a side chain, e.g., glycyl; or (e) by increasing the number of sulfation or glycosylation.

[0087] As noted above, the kits also can include controls or references. Examples of a positive control include, but are not limited to, normal striated/skeletal muscle cells, cardiac cells, and cells from a positively-identified individual having a tumor having a skeletal muscle origin including those listed above. The positive controls preferably display cytoplasmic expression of at least one of the myoblastic differentiation biomarkers, although it is contemplated in some instances that the positive control can display nuclear expression of the biomarker. Likewise, examples of a negative control include, but are not limited to, any non-striated or non-skeletal muscle cell. Positive and/or negative controls can be used to validate the activity and correct usage of reagents employed in accordance with the invention. Controls can include samples, such as tissue sections, cells fixed on glass slides, RNA preparations from tissues or cell lines, and the like, known to be either positive or negative for the presence of biomarker, especially p63. The design and use of controls is standard and well within the routine capabilities of one of skill in the art.

[0088] The kits also can include a means for processing a biological sample or for disrupting cell structures in a biological sample so as to expose intracellular nucleic acid and amino acid molecules. Examples of such means are described in greater detail below.

[0089] The kits also can include a package insert describing procedures for carrying out any one of the method described herein or analytical information for correlating the level of biomarker expression measured with an individual's likelihood of having developed a tumor having a skeletal muscle origin or the likely prognosis of an individual already diagnosed with such a tumor. Likewise, the package insert can include representative images of tumor samples with low or high levels of biomarker expression as compared to an appropriate control. The kits can be promoted, distributed or sold as units for performing the methods described below.

[0090] The kits also can include a receptacle or other means for capturing a sample to be evaluated for the biomarker, and means for determining the presence and/or quantity of the biomarker in the sample.

[0091] The kits also can include reagents for detecting or determining myoblastic differentiation biomarker expression. Examples of reagents include, but are not limited to, fluorescent tags {e.g., fluorescein, rhodamine, especially the Alexa Fluor^® family of fluorescent dyes available from Invitrogen/Molecular Probes), radiolabel tags, enzymatic tags {e.g., biotin/avidin, alkaline phosphatase, etc.) or other tags. The reagents also can include, for example, secondary or tertiary antibodies or reagents for enzymatic reactions, where the enzymatic reactions produce a product that can be visualized. Alternatively, the reagents can include, for example, an agent for processing a biological sample for histological staining {e.g., Hematoxylin & Eosin) and immunohistochemistry.

[0092] The kits also can include at least one buffer. Examples of buffers include, but are not limited to, cell isolation buffers, fixation buffers, lysis buffers, permeabilization buffers, sonication buffers, separation buffers, stabilization buffers and wash buffers.

[0093] Any or all of the kit components can be provided within containers that protect them from the external environment, such as in sealed containers.

[0094] The kits therefore can be for detecting, diagnosing, differentiating, prognosing and monitoring a tumor via myoblastic differentiation biomarkers at the nucleic acid level. Such kits are compatible with both manual and automated nucleic acid detection techniques {e.g., gene arrays, Northern blotting or Southern blotting). These kits can include a plurality of probes, for example, from two to thirty nucleic acid probes that specifically bind to distinct biomarkers, fragments or variants thereof. Alternatively, the kits can contain at least two probes, at least three probes, at least four probes, at least five probes, at least six probes, at least seven probes, at least eight probes, at least nine probes, at least ten probes, at least eleven probes, at least twelve probes, at least thirteen probes, at least fourteen probes, at least fifteen probes, at least sixteen probes, at least seventeen probes, at least eighteen probes, at least nineteen probes, at least twenty probes, at least twenty-five probes, or at least thirty probes. Each probe can be provided in the kit as an individual reagent or, alternatively, as a cocktail comprising the selected number of probes directed to the same or different biomarkers.

[0095] Likewise, the kits can be for detecting, diagnosing, differentiating, prognosing and monitoring a tumor with biomarkers at the amino acid level. Such kits are compatible with both manual and automated immunohistochemistry techniques {e.g., cell staining, ELISA or Western blotting). These kits can include a plurality of probes, for example, from two to thirty antibodies that specifically bind to distinct biomarkers, fragments or variants thereof. Alternatively, the kits can contain at least two antibodies, at least three antibodies, at least four antibodies, at least five antibodies, at least six antibodies, at least seven antibodies, at least eight antibodies, at least nine antibodies, at least ten antibodies, at least eleven antibodies, at least twelve antibodies, at least thirteen antibodies, at least fourteen antibodies, at least fifteen antibodies, at least sixteen antibodies, at least seventeen antibodies, at least eighteen antibodies, at least nineteen antibodies, at least twenty antibodies, at least twenty-five antibodies or at least thirty antibodies. Each antibody can be provided in the kit as an individual reagent or, alternatively, as an antibody cocktail comprising the selected number of antibodies directed to the same or different biomarkers.

[0096] The kits therefore can include nucleotide probes and/or antibodies to detect myoblastic differentiation biomarkers such as p63, desmin, myogenin, MyoD1 and myoglobin.

[0097] Alternatively, the kits can include nucleotide probes and/or antibodies to detect p63, desmin, myogenin and MyoD1 .

[0098] Alternatively, the kits can include nucleotide probes and/or antibodies to detect p63, desmin, myogenin and myoglobin.

[0099] Alternatively, the kits can include nucleotide probes and/or antibodies to detect p63, desmin, MyoD1 and myoglobin.

[00100] Alternatively, the kits can include nucleotide probes and/or antibodies to detect p63, myogenin, MyoD1 and myoglobin.

[00101] Alternatively, the kits can include nucleotide probes and/or antibodies to detect p63, desmin and myogenin.

[00102] Alternatively, the kits can include nucleotide probes and/or antibodies to detect p63, desmin and MyoD1 .

[00103] Alternatively, the kits can include nucleotide probes and/or antibodies to detect p63, desmin and myoglobin.

[00104] Alternatively, the kits can include nucleotide probes and/or antibodies to detect p63, myogenin and MyoD1 .

[00105] Alternatively, the kits can include nucleotide probes and/or antibodies to detect p63, myogenin and myoglobin.

[00106] Alternatively, the kits can include nucleotide probes and/or antibodies to detect p63, MyoD1 and myoglobin.

[00107] Alternatively, the kits can include nucleotide probes and/or antibodies to detect p63 and desmin. [00108] Alternatively, the kits can include nucleotide probes and/or antibodies to detect p63 and myogenin.

[00109] Alternatively, the kits can include nucleotide probes and/or antibodies to detect p63 and MyoD1 .

[00110] Alternatively, the kits can include nucleotide probes and/or antibodies to detect p63 and myoglobin.

[00111] Alternatively, the kits can include nucleotide probes and/or antibodies to detect p63 only.

[00112] Methods

[00113] Biological Sample Collection and Processing

[00114] In some instances, a prerequisite to performing the detecting, diagnosing, differentiating, prognosing or monitoring methods may be collecting and processing a biological sample from an individual. As such, the methods generally begin by collecting a biological sample from an individual having or suspected of having a tumor.

[00115] As used herein, "biological sample" means any collection of cells, tissues, organs or bodily fluids in which expression of at least one myoblastic differentiation biomarker can be detected. Examples of suitable biological samples include, but are not limited to, biopsy specimens of cells, tissues or organs, bodily fluids and smears. Biopsy specimens can be obtained by a variety of techniques including, but not limited to, scraping or swabbing an area, using a needle to aspirate cells or bodily fluids, or removing a tissue sample. When the sample is a bodily fluid, it can include, but is not limited to, blood, lymph, urine, saliva, aspirates or any other bodily secretion or derivative thereof. When the sample is blood, it can include whole blood, plasma, serum or any derivative of blood.

[00116] As such, the biological sample can be any sample that is suspected of containing a nucleic acid molecule encoding the at least one myoblastic differentiation biomarker or the biomarker itself, such as a tumor tissue biopsy section or specimen, a homogenized tumor tissue extract, an isolated cell, a cytosolic preparation, a cell membrane preparation, separated or purified forms of any of the above compositions, or even any biological fluid that may contact such tissues, including blood and lymphatic fluid.

[00117] Because tumors having a skeletal muscle origin can arise in virtually every site of the body, it is intended that the biological sample can come from any part of the body having or suspected of having such a tumor, especially a soft tissue tumor. Common examples of biological samples for use herein include, but are not limited to, soft tissues and the cytoskeletal system, head and neck samples {e.g., sinuses, middle ear, nares and throat), genitourinary tract samples (e.g., bladder, testicle, prostate, urethra, vulva, vagina, cervix and uterus), orbital sample, parameningeal sample.

[00118] Methods of collecting and processing [e.g., fixing, isolating, staining, etc.) a biological sample are well known in the art. See, e.g., Holland et al. (2003) Mutat. Res. 543:217-234; Tworoger & Hankison (2006) Cancer Epidemiol. Biomarkers Prev. 15:1578-1581 . See also, "Technical and Operational Best Practices: Biospecimen Collection, Processing, Storage, Retrieval, and Dissemination," available on the World Wide Web at biospecimens.cancer.gov/bestpractices/to/bcpsrd. asp.

[00119] Fixative and staining solutions can be applied to, for example, cells or tissues for preserving them and for facilitating examination. Body samples, particularly tissue samples, can be transferred to a glass slide for viewing under magnification. For example, the body sample can be a formalin-fixed, paraffin-embedded tissue sample, particularly a primary tumor sample.

[00120] As used herein, "individual" means a mammal such as laboratory animal (including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, etc.), domestic animal (including, but not limited to, dogs and cats), farm animal (including, but not limited to, sheep, goats, pigs, horses and cows), and human and non-human primates.

[00121] Many factors must be considered at the initial collection stage including, but not limited to, tissue type, time of collection, container used, preservatives and other additives, transport means and length of transit time. These factors all can affect the quality of the biological samples and the stability of the biomarker. [00122] Detecting and Diagnosing Methods

[00123] Methods of the invention can include detecting myoblastic differentiation in cells suspected of undergoing myoblastic differentiation. The method can include detecting, measuring or observing expression of at least one myoblastic differentiation biomarker in a biological sample such as cells, where cytoplasmic expression of the at least one biomarker in the cells indicates that the cells are undergoing myoblastic differentiation (i.e., are malignant cells).

[00124] Methods of the invention also include diagnosing an individual as having a tumor having a skeletal muscle origin. The method can include detecting, measuring or observing cytoplasmic expression of at least one myoblastic differentiation biomarker in cells from a biological sample from the individual, where cytoplasmic expression of the at least one biomarker in the cells indicates that individual has the tumor having a skeletal muscle origin.

[00125] In either method, the biological sample can be processed as described above.

[00126] As used herein, "myogenic differentiation," "myoblastic differentiation," "striated muscle differentiation," "skeletal muscle differentiation" and "rhabdomyoblastic differentiation" are used interchangeably to mean malignant cells undergoing muscle differentiation, although the latter two terms are intended to be associated more with a pathological process than the first term. As such, these terms are intended to exclude cells undergoing normal myogenic differentiation.

[00127] One can use any method available for detecting expression of a polynucleotide and polypeptide myoblastic differentiation biomarker such as p63. As used herein, "detecting expression" means determining the quantity or presence of a biomarker polynucleotide or its polypeptide expression product. As such, detecting expression encompasses instances where a biomarker is determined not to be expressed, not to be detectably expressed, expressed at a low level, expressed at a normal level or overexpressed.

[00128] Expression of a biomarker can be determined by normalizing the level of a reference marker/control, which can be all measured transcripts (or their products) in the sample or a particular reference set of RNA transcripts (or their products). Normalization can be performed to correct for or normalize away both differences in the amount of biomarker assayed and variability in the quality of the biomarker type used. Therefore, an assay typically measures and incorporates the expression of certain normalizing polynucleotides or polypeptides, including well-known housekeeping genes, such as, for example, GAPDH and/or actin. Alternatively, normalization can be based on the mean or median signal of all of the assayed biomarkers or a large subset thereof (global normalization approach).

[00129] To determine overexpression, the sample can be compared with a corresponding sample that originates from, for example, a healthy individual. That is, the "normal" level of expression is the level of expression of the biomarker in, for example, a tissue sample from an individual not afflicted with a tumor. Such a sample can be present in standardized form. Sometimes, determining biomarker overexpression requires no comparison between the sample and a corresponding sample that originated from a healthy individual. For example, detecting overexpression of a biomarker indicative of a poor prognosis in a tumor sample may preclude the need for comparison to a corresponding sample that originates from a healthy individual. Moreover, no expression, underexpression or normal expression (i.e., the absence of overexpression) of a biomarker or combination of biomarkers of interest provides useful information regarding the prognosis of an individual.

[00130] Methods of detecting and quantifying polynucleotide biomarkers in a sample are well known in the art. Such methods include, but are not limited to gene expression profiling, which are based on hybridization analysis of polynucleotides, and sequencing of polynucleotides. The most commonly used methods for detecting and quantifying polynucleotide expression include Northern blotting and in situ hybridization (Parker & Barnes (1999) Methods Mol. Biol. 106:247-283), RNAse protection assays (Hod (1992) Biotechniques 13:852-854), PCR-based methods, such as RT-PCR (Weis et al. (1992) TIG 8:263-264), and array-based methods (Schena et al. (1995) Science 270:467-470). Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes, or DNA- protein duplexes in, for example, an oligonucleotide-linked immunosorbent assay ("OLISA"). See, Lee et al. (1985) FEBS Lett. 190:120-124; Han et al. (2010) Bioconjug. Chem. 21 :2190-2196; Miura et al. (1987) Biochem. Biophys. Res. Commun. 144:930- 935; and Tanha & Lee (1997) Nucleic Acids Res. 25:1442-1449. Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression ("SAGE") and gene expression analysis by massively parallel signature sequencing. See, Velculescu et al. (1995) Science 270: 484-487.

[00131] Isolated RNA can be used to determine the level of biomarker transcripts (i.e., mRNA) in a sample, as many expression detection methods use isolated RNA. The starting material typically is total RNA isolated from a body sample, such as a tumor or tumor cell line, and corresponding normal tissue or cell line, respectively. Thus, RNA can be isolated from a variety of primary tumors, including breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis, ovary, uterus, and the like, or tumor cell lines. If the source of mRNA is a primary tumor, mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g., formalin-fixed) tissue samples.

[00132] Methods of isolating polynucleotides such as RNA from a sample are well known in the art. See, e.g., Molecular Cloning: A Laboratory Manual, 3^rd ed. (Sambrook et al. eds., Cold Spring Harbor Press 2001 ); and Current Protocols in Molecular Biology (Ausubel et al. eds., John Wiley & Sons 1995). Methods for RNA extraction from paraffin-embedded tissues also are well known in the art. See, e.g., Rupp & Locker (1987) Lab. Invest. 56:A67; and De Andres et al. (1995) Biotechniques 18:42-44. Moreover, isolation/purification kits are commercially available for isolating polynucleotides such as RNA (Qiagen; Valencia, CA). For example, total RNA from cells in culture can be isolated using Qiagen RNeasy® Mini-Columns. Other commercially available RNA isolation/purification kits include MasterPure™ Complete DNA and RNA Purification Kit (Epicentre; Madison, Wl.) and Paraffin Block RNA Isolation Kit (Ambion; Austin, TX). Total RNA from tissue samples can be isolated, for example, using RNA Stat-60 (Tel-Test; Friendswood, TX). RNA prepared from a tumor can be isolated, for example, by cesium chloride density gradient centrifugation. Additionally, large numbers of tissue samples can be processed using techniques well known in the art, such as, for example, the single-step RNA isolation process of Chomczynski (US Patent No. 4,843,155). [00133] Once isolated, the polynucleotide, such as mRNA, can be used in hybridization or amplification assays including, but not limited to, Southern or Northern blotting, PCR and probe arrays. One method of detecting polynucleotide levels involves contacting the isolated polynucleotides with a nucleic acid molecule (probe) that can hybridize to the desired polynucleotide target. The nucleic acid probe can be, for example, a full-length DNA, or a portion thereof, such as an oligonucleotide of at least about 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400 or 500 nucleotides or more in length and sufficient to specifically hybridize under stringent conditions to a polynucleotide such as an mRNA or genomic DNA encoding a biomarker of interest. Hybridization of a polynucleotide encoding the biomarker of interest with the probe indicates that the biomarker in question is being expressed.

[00134] Stringent hybridization conditions are defined as hybridizing at 68°C in 5x SSC/5x Denhardt's solution/1 .0% SDS, and washing in 0.2x SSC/0.1 % SDS +/- 100 g/ml denatured salmon sperm DNA at room temperature (RT), and moderately stringent hybridization conditions are defined as washing in the same buffer at 42°C. Additional guidance regarding such conditions is readily available in the art, for example, in Molecular Cloning: A Laboratory Manual, 3^rd ed. (Sambrook et al. eds., Cold Spring Harbor Press 2001 ); and Current Protocols in Molecular Biology (Ausubel et al. eds., John Wiley & Sons 1995).

[00135] Another method of detecting polynucleotide expression levels involves immobilized polynucleotides on a solid surface and contacting the immobilized polynucleotides with a probe, for example by running isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. Alternatively, the probes can be immobilized on a solid surface and isolated mRNA is contacted with the probes, for example, in an Agilent Gene Chip Array.

[00136] For example, microarrays can be used to detect polynucleotide expression. Microarrays are particularly well suited because of the reproducibility between different experiments. DNA microarrays provide one method for the simultaneous measurement of the expression levels of large numbers of polynucleotides. Each array consists of a reproducible pattern of capture probes attached to a solid support. Labeled RNA or DNA is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative gene expression levels. See, e.g., US Patent Nos. 6,040,138; 5,800,992; 6,020,135; 6,033,860 and 6,344,316. High- density oligonucleotide arrays are particularly useful for determining expression profiles for a large number of polynucleotides in a sample.

[00137] Methods of synthesizing these arrays using mechanical synthesis methods are described in, for example, US Patent No. 5,384,261 . Although a planar array surface generally is used, the array can be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays can be nucleic acids (or peptides) on beads, gels, polymeric surfaces, fibers (such as fiber optics), glass or any other appropriate substrate. See, e.g., US Patent Nos. 5,770,358; 5,789,162; 5,708,153; 6,040,193 and 5,800,992.

[00138] As such, PCR-amplified inserts of cDNA clones can be applied to a substrate in a dense array. For example, at least about 10,000 nucleotide sequences can be applied to the substrate. The microarrayed genes, immobilized on the microchip at 10,000 elements each, are suitable for hybridization under stringent conditions. Fluorescently labeled cDNA probes can be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance.

[00139] With dual color fluorescence, separately labeled cDNA probes generated from two sources of polynucleotide can be hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified molecule is thus determined simultaneously. The miniaturized scale of the hybridization affords a convenient and rapid evaluation of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels. See, Schena et al. (1996) Proc. Natl. Acad. Sci. USA 93:106-149. Advantageously, microarray analysis can be performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix® GenChip Technology, or Agilent® Ink-Jet Microarray Technology. The development of microarray methods for large-scale analysis of gene expression makes it possible to search systematically for molecular markers of cancer classification and outcome prediction in a variety of tumor types.

[00140] Another method of detecting polynucleotide expression levels involves a digital technology developed by NanoString® Technologies (Seattle, WA) and based on direct multiplexed measurement of gene expression, which offers high levels of precision and sensitivity (<1 copy per cell). The method uses molecular "barcodes" and single molecule imaging to detect and count hundreds of unique transcripts in a single reaction. Each color-coded barcode is attached to a single target-specific probe corresponding to a gene of interest. Mixed together with controls, they form a multiplexed CodeSet. Two -50 base probes per mRNA can be included for hybridization. The reporter probe carries the signal, and the capture probe allows the complex to be immobilized for data collection. After hybridization, the excess probes are removed and the probe/target complexes aligned and immobilized in an nCounter® Cartridge. Sample cartridges are placed in a digital analyzer for data collection. Color codes on the surface of the cartridge are counted and tabulated for each target molecule.

[00141] Another method of detecting polynucleotide expression levels involves nucleic acid amplification, for example, by RT-PCR (US Patent No. 4,683,202), ligase chain reaction (Barany (1991 ) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1 173-1 177), Q-Beta Replicase (Lizardi et al., (1988) Bio/Technology 6:1 197), rolling circle replication (US Patent No. 5,854,033), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known in the art. Likewise, biomarker expression can be assessed by quantitative fluorogenic RT-PCR (i.e., the TaqMan® System). For PCR analysis, methods and software are available to determine primer sequences for use in the analysis. These methods are particularly useful for detecting polynucleotides present in very low numbers.

[00142] Additional methods of detecting polynucleotide expression levels of RNA may be monitored using a membrane blot (such as used in hybridization analysis such as Northern or Southern blotting, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See, e.g., US Patents Nos. 5,770,722; 5,874,219; 5,744,305; 5,677,195 and 5,445,934. Polynucleotide biomarker expression also can include using nucleic acid probes in solution.

[00143] Another method of detecting polynucleotide expression levels involves SAGE, which is a method that allows the simultaneous and quantitative analysis of a large number of polynucleotides without the need of providing an individual hybridization probe for each transcript. First, a short sequence tag (about 10-14 bp) is generated that contains sufficient information to uniquely identify a transcript, provided that the tag is obtained from a unique position within each transcript. Then, many transcripts are linked together to form long serial molecules that can be sequenced, revealing the identity of the multiple tags simultaneously. The expression pattern of any population of transcripts can be quantitatively evaluated by determining the abundance of individual tags and identifying the gene corresponding to each tag. See, Velculescu et al. (1995), supra.

[00144] Another method of detecting polynucleotide expression levels involves massively parallel signature sequencing ("MPSS"). See, Brenner et al. (2000) Nat. Biotech. 18:630-634. This sequencing combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate diameter microbeads. First, a microbead library of DNA templates can be constructed by in vitro cloning. This is followed by assembling a planar array of the template-containing microbeads in a flow cell at a high density (typically greater than 3.0 x 10⁶ microbeads/cm²). The free ends of the cloned templates on each microbead are analyzed simultaneously, using a fluorescence-based signature sequencing method that does not require DNA fragment separation. This method has been shown to simultaneously and accurately provide, in a single operation, hundreds of thousands of gene signature sequences from a yeast DNA library.

[00145] Likewise, methods of detecting and quantifying polypeptides in a sample are well known in the art and include, but are not limited to, immunohistochemistry and proteomics-based methods.

[00146] For example, a tissue sample can be collected by, for example, biopsy techniques known in the art. Samples can be frozen for later preparation or immediately placed in a fixative solution. Tissue samples can be fixed by treatment with a reagent, such as formalin, gluteraldehyde, methanol, and the like, and embedded in paraffin. Methods for preparing slides for immunohistochemical analysis from formalin- fixed, paraffin-embedded tissue samples are well known in the art.

[00147] Some samples may need to be subjected to antigen retrieval or antigen unmasking to make the biomarker polypeptides accessible to, for example, antibody binding. As used herein, "antigen retrieval" or "antigen unmasking" means methods for increasing antigen accessibility or recovering antigenicity in, for example, formalin-fixed, paraffin -em bedded tissue samples. Formalin fixation of tissue samples results in extensive cross-linking of proteins that can lead to the masking or destruction of antigen sites and, subsequently, poor antibody staining. Any method of making antigens more accessible for antibody binding may be used in the practice of the invention, including those antigen retrieval methods known in the art. See, e.g., Tumor Marker Protocols (Hanausek & Walaszek, eds., Humana Press, Inc. 1988); and Shi et al., Antigen Retrieval Techniques: Immunohistochemistry and Molecular Morphology (Eaton Publishing 2000).

[00148] Methods of antigen retrieval are well known in the art. Examples of such methods include, but are not limited to, treatment with proteolytic enzymes {e.g., trypsin, chymotrypsin, pepsin, pronase and the like) or antigen retrieval solutions. Antigen retrieval solutions can include citrate buffer, pH 6.0, Tris buffer, pH 9.5, EDTA, pH 8.0, L.A.B. ("Liberate Antibody Binding Solution"; Polysciences; Warrington, PA), antigen retrieval Glyca solution (Biogenex; San Ramon, CA), citrate buffer solution, pH 4.0, Dawn® detergent (Proctor & Gamble; Cincinnati, OH), deionized water and 2% glacial acetic acid. Such an antigen retrieval solutions can be applied to a formalin-fixed tissue sample and then heated in an oven (e.g., at 60°C), steamed (e.g., at 95°C) or pressure cooked (e.g., at 120°C) for a pre-determined time periods. Alternatively, antigen retrieval can be performed at room temperature. As such, incubation times will vary with the particular antigen retrieval solution selected and with the incubation temperature. For example, an antigen retrieval solution can be applied to a sample for as little as about 5, 10, 20 or 30 minutes or up to overnight. The design of assays to determine the appropriate antigen retrieval solution and optimal incubation times and temperatures is standard and well within the routine capabilities of one of skill in the art.

[00149] Following antigen retrieval, samples are blocked using an appropriate blocking agent (e.g., hydrogen peroxide). An antibody directed to a biomarker of interest then is incubated with the sample for a time sufficient to permit antigen-antibody binding. As described elsewhere, at least one antibody directed to p63 can be used to detect the tumor. Where more than one antibody may be used, these antibodies can be added to a single sample sequentially as individual antibody reagents, or simultaneously as an antibody cocktail. Alternatively, each individual antibody can be added to a separate tissue section from a single biological sample, and the resulting data pooled.

[00150] Methods of detecting antibody binding are well known in the art. Antibody binding to a biomarker of interest can be detected through the use of chemical reagents that generate a detectable signal that corresponds to the level of antibody binding, and, accordingly, to the level of biomarker protein expression. For example, antibody binding can be detected through the use of a secondary antibody that is conjugated to a labeled polymer. Examples of labeled polymers include but are not limited to polymer-enzyme conjugates. The enzymes in these complexes are typically used to catalyze the deposition of a chromogen at the antigen-antibody binding site, thereby resulting in cell or tissue staining that corresponds to expression level of the biomarker of interest. Enzymes of particular interest include horseradish peroxidase (HRP) and alkaline phosphatase (AP). Commercially antibody detection systems include, for example, the Dako Envision+system (Glostrup; Denmark) and Biocare Medical's Mach 3 System (Concord, CA), and can be used herein. [00151] Detecting antibody binding can be facilitated by coupling the antibody to a detectable moiety. Examples of detectable moieties include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, galactosidase and acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriaziny-lamine fluorescein, dansyl chloride and phycoerythrin. An example of a luminescent material is luminol. Examples of bioluminescent materials include luciferase, luciferin and aequorin. Examples of radioactive materials include ¹²⁵l, ¹³¹1, ³⁵S and ³H. See also, US Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241 .

[00152] In regard to additional antibody detection methods, there also exists video microscopy and software methods for quantitatively determining an amount of multiple molecular species {e.g., biomarker proteins) in a biological sample, where each molecular species present is indicated by a representative dye marker having a specific color. Such methods are known in the art as a colorimetric analysis method. In these methods, video-microscopy is used to provide an image of the biological sample after it has been stained to visually indicate the presence of a particular biomarker of interest. See, e.g., US Patent Nos. 7,065,236 and 7,133,547, which disclose the use of an imaging system and associated software to determine the relative amounts of each molecular species present based on the presence of representative color dye markers as indicated by those color dye markers' optical density or transmittance value, respectively, as determined by an imaging system and associated software. These methods provide quantitative determinations of the relative amounts of each molecular species in a stained biological sample using a single video image that is "deconstructed" into its component color parts.

[00153] Polynucleotide and polypeptide myoblastic differentiation biomarkers for use in detecting and diagnosing tumors having a skeletal muscle origin can include p63, desmin, myogenin, MyoD1 and myoglobin. [00154] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin, myogenin and MyoD1 .

[00155] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin, myogenin and myoglobin.

[00156] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin, MyoD1 and myoglobin.

[00157] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, myogenin, MyoD1 and myoglobin.

[00158] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin and myogenin.

[00159] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin and MyoD1 .

[00160] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin and myoglobin.

[00161] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, myogenin and MyoD1 .

[00162] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, myogenin and myoglobin.

[00163] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, MyoD1 and myoglobin.

[00164] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 and desmin.

[00165] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 and myogenin.

[00166] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 and MyoD1 .

[00167] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 and myoglobin.

[00168] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 only. [00169] With respect to detecting myoblastic differentiation or diagnosing an individual, preferred biomarkers include at least p63, where cytoplasmic expression of p63 indicates myoblastic differentiation. p63 preferably can be detected at the amino acid/protein level because cytoplasmic expression of p63 is being assessed. As such, one of skill in the art typically would use ELISA, IP, IF, flow cytometry or IHC to examine cytoplasmic p63 expression, preferably IHC. As used herein, "immunohistochemistry" or "IHC" means detecting antigens {e.g., p63) in cells of a tissue section with antibodies or functional fragments thereof that bind specifically to antigens in the biological sample.

[00170] Differentiating Methods

[00171] Methods of the invention can include differentiating tumors having a skeletal muscle origin from tumors that do not have a skeletal muscle origin. The method can include detecting, measuring or observing cytoplasmic expression of at least one myoblastic differentiation biomarker in cells from a biological sample suspected of having a tumor having a skeletal muscle origin, where cytoplasmic expression of the at least one biomarker indicates that the tumor has a skeletal muscle origin, or where lack of cytoplasmic expression of the at least one biomarker indicates that the tumor does not have a skeletal muscle origin. Tumors that do not have a skeletal muscle origin may have nuclear expression of the biomarker or no biomarker expression.

[00172] In the method, the biological sample can be processed as described above. Likewise, the at least one myoblastic differentiation biomarker can be examined at the nucleic acid or amino acid level as described above.

[00173] Polynucleotide and polypeptide myoblastic differentiation biomarkers for use in differentiating a tumor having a skeletal muscle origin from one that does not have a skeletal muscle origin can include p63, desmin, myogenin, MyoD1 and myoglobin.

[00174] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin, myogenin and MyoD1 .

[00175] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin, myogenin and myoglobin.

[00176] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin, MyoD1 and myoglobin. [00177] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, myogenin, MyoD1 and myoglobin.

[00178] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin and myogenin.

[00179] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin and MyoD1 .

[00180] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin and myoglobin.

[00181] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, myogenin and MyoD1 .

[00182] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, myogenin and myoglobin.

[00183] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, MyoD1 and myoglobin.

[00184] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 and desmin.

[00185] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 and myogenin.

[00186] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 and MyoD1 .

[00187] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 and myoglobin.

[00188] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 only.

[00189] With respect to differentiating tumors, preferred biomarkers include at least p63, where cytoplasmic expression of p63 indicates that the tumor has a skeletal muscle origin. Greater or more prevalent levels of cytoplasmic p63 expression generally indicate that the tumor has a skeletal muscle origin, whereas lesser of diffuse levels of cytoplasmic p63 expression, nuclear p63 expression or no p63 expression generally indicate that the tumor does not have a skeletal muscle origin. p63 preferably can be detected at the amino acid/protein level because cytoplasmic expression of p63 is being assessed. As such, one of skill in the art typically would use ELISA, IP, IF, flow cytometry or IHC to examine cytoplasmic p63 expression, preferably IHC.

[00190] Prognosing Methods

[00191] Methods of the invention can include prognosing an individual having or suspected of having a tumor having a skeletal muscle origin. The method can include detecting, measuring or observing cytoplasmic expression of at least one myoblastic biomarker in cells from the individual, where elevated cytoplasmic expression of the biomarker in cells (or alternatively an increased number of cells having cytoplasmic expression of the biomarker) when compared to a control or reference indicates a poor prognosis, or where attenuated cytoplasmic expression of the biomarker in cells (or alternatively a decreased number of cells having cytoplasmic expression of the biomarker) when compared to a control or reference indicates a good/favorable prognosis. Altered cytoplasmic expression of at least p63 can be used to indicate tumor prognosis (i.e., poor or good/favorable prognosis). As such, altered expression of a particular biomarker or combination of biomarkers permits the differentiation of individuals having a tumor that is likely to experience recurrence and/or metastasis (i.e., poor prognosis) from those who are more likely to remain tumor free (i.e., good/favorable prognosis).

[00192] As used herein, "prognose," "prognoses," "prognosis" and "prognosing" means predictions about or predicting a likely course or outcome of a disease or disease progression, particularly with respect to a likelihood of, for example, disease remission, disease relapse, tumor recurrence, metastasis and death (i.e., the outlook for chances of survival). As used herein, "good prognosis" or "favorable prognosis" means a likelihood that an individual having a tumor, particularly a tumor having a skeletal muscle origin, will remain disease-free (i.e., tumor-free). As used herein, "poor prognosis" means a likelihood of a relapse or recurrence of the underlying cancer or tumor, metastasis or death. Individuals classified as having a good prognosis remain free of the underlying cancer or tumor. Conversely, individuals classified as having a bad prognosis experience disease relapse, tumor recurrence, metastasis or death. [00193] One of skill in the art is familiar with the time frame(s) for assessing prognosis and outcome. Examples of such time frames include, but are not limited to, less than one year, about one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more years. With respect to tumors, the relevant time for assessing prognosis or disease-free survival time often begins with the surgical removal of the tumor or suppression, mitigation or inhibition of tumor growth. Thus, for example, a good prognosis can be likelihood that the individual having the tumor will remain free of the underlying cancer or tumor for a period of at least about five, more particularly, a period of at least about ten years. In contrast, for example, a bad prognosis can be likelihood that the individual having the tumor experiences disease relapse, tumor recurrence, metastasis or death within a period of less than about five years, more particularly a period of less than about ten years. Methods of prognosing cancer are well known in the art and described above.

[00194] The biomarkers of interest can be statistically significant for assessment of the likelihood of tumor recurrence or death due to the underlying disease. Methods for assessing statistical significance are well known in the art and include, for example, using a log-rank test, Cox analysis and Kaplan-Meier curves. A p-value of less than 0.05 can be used to constitute statistical significance.

[00195] The expression level of at least one biomarker in a biological sample can be indicative of a poor prognosis and thereby used to identify individuals who are more likely to suffer a recurrence of the underlying tumor. The therefore methods involve detecting the expression levels of at least one biomarker in a biological sample that is indicative of the disease.

[00196] In some instances, overexpression of a biomarker or combination of biomarkers of interest in a sample can be indicative of a poor prognosis. As used herein, "indicative of a poor prognosis" means an altered expression of particular biomarkers or combination of biomarkers is associated with an increased likelihood of relapse or recurrence of the underlying cancer or tumor, metastasis or death. For example, "indicative of a poor prognosis" may refer to an increased likelihood of relapse or recurrence of the underlying cancer or tumor, metastasis, or death within ten years, such as five years. [00197] In other instances, the absence of overexpression of a biomarker or combination of biomarkers of interest is indicative of a good prognosis. As used herein, "indicative of a good prognosis" means an increased likelihood that one will remain cancer free. "Indicative of a good prognosis" also means an increased likelihood that one will remain cancer-free for ten years, such as five years.

[00198] In the method, the biological sample can be processed as described above. Likewise, the at least one myoblastic differentiation biomarker can be examined at the nucleic acid or amino acid level as described above.

[00199] The method also can include morphologically evaluating cells under a microscope {e.g., electron microscopy or light microscopy) for indicators to confirm the prognosis. Examples of indicators include, but are not limited to, cross-striations by light microscopy, actin and myosin filaments, and Z bands.

[00200] The method also can include selecting or adjusting an appropriate therapy in an individual based upon whether there is a poor prognosis {e.g., initiating a therapy, adding a therapy, increasing a therapy and/or switching a therapy) or a good/favorable prognosis {e.g., initiating a therapy, removing a therapy, decreasing a therapy and/or switching a therapy).

[00201] Polynucleotide and polypeptide myoblastic differentiation biomarkers for use in prognosing an individual having a tumor having a skeletal muscle origin can include p63, desmin, myogenin, MyoD1 and myoglobin.

[00202] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin, myogenin and MyoD1 .

[00203] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin, myogenin and myoglobin.

[00204] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin, MyoD1 and myoglobin.

[00205] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, myogenin, MyoD1 and myoglobin.

[00206] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin and myogenin. [00207] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin and MyoD1 .

[00208] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin and myoglobin.

[00209] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, myogenin and MyoD1 .

[00210] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, myogenin and myoglobin.

[00211] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, MyoD1 and myoglobin.

[00212] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 and desmin.

[00213] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 and myogenin.

[00214] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 and MyoD1 .

[00215] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 and myoglobin.

[00216] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 only.

[00217] With respect to prognosing an individual, preferred biomarkers include at least p63, where cytoplasmic expression of p63 indicates the prognosis. Greater or more prevalent levels of cytoplasmic p63 expression (or greater number of expressing cells) generally indicate a poor prognosis, whereas lesser or diffuse levels of cytoplasmic p63 expression (or decreased number of expressing cells) generally indicate a good/favorable prognosis. p63 preferably can be detected at the amino acid/protein level because cytoplasmic expression of p63 is being assessed. As such, one of skill in the art typically would use ELISA, IP, IF, flow cytometry or IHC to examine cytoplasmic p63 expression, preferably IHC. [00218] Monitoring Methods

[00219] Methods of the invention can include monitoring progression of a tumor having a skeletal muscle origin in an individual having the tumor. The method can include detecting, measuring or observing cytoplasmic expression of at least one myoblastic differentiation biomarker in cells from first and second chronological biological samples from the individual, where elevated cytoplasmic expression of the at least one biomarker in cells (or alternatively an increased number of cells having cytoplasmic expression of the biomarker) of the second chronological sample when compared to cytoplasmic expression of the at least biomarker in cells (or alternatively number of cells having cytoplasmic expression of the biomarker) of the first chronological sample indicates tumor progression, or where decreased cytoplasmic expression of the at least one biomarker in cells (or alternatively a decreased number of cells having cytoplasmic expression of the biomarker) of the second chronological sample when compared to cytoplasmic expression of the at least one biomarker in cells (or alternatively number of cells having cytoplasmic expression of the biomarker) of the first chronological sample indicates tumor regression.

[00220] Methods of the invention also include or monitoring efficacy of a therapy in an individual having a tumor having a skeletal muscle origin. The method can include detecting, measuring or observing cytoplasmic expression of at least one myoblastic differentiation biomarker in cells from at least two chronological biological samples from the individual, where elevated cytoplasmic expression of the at least one biomarker in cells in a later-obtained chronological sample indicates an ineffectiveness of therapy when compared to cytoplasmic expression of the at least biomarker in cells in an earlier-obtained chronological sample, or where attenuated cytoplasmic expression of the at least one biomarker in cells in a later-obtained chronological sample indicates effectiveness of therapy when compared to cytoplasmic expression of the at least biomarker in cells in an earlier-obtained chronological sample.

[00221] Either method can be used to detect minimal residual disease and/or relapse and/or remission following therapy.

[00222] As with the other methods described above, the monitoring methods include examining cytoplasmic expression of at least one biomarker. Unlike the methods above, where typically only a single biological sample is examined for cytoplasmic biomarker expression, the monitoring methods include examining at least two chronological samples from the individual, particularly where at least one sample is from after a treatment or therapy has been initiated. As used herein, "chronological" or "chronologically," with respect to the timing of obtaining samples, means within an order of events, although not necessarily immediately successive, but that at least one sample was obtained at a point prior to another.

[00223] As such, establishing the declining presence or absence of the at least one myoblastic differentiation biomarker in a biological sample or series of biological samples from an individual may be used to evaluate the efficacy of a therapy for the eradication of a tumor having a skeletal muscle origin. Conversely, the presence of the at least one myoblastic biomarker in the biological samples can be used as an indicator that the tumor may be resistant to the therapy, and/or of the presence of minimal residual disease or disease relapse. Indications of any of the above provide a clinician with information critical in the treatment of the individual and can be used to assess the potential for continued or different therapeutic intervention modalities.

[00224] In either method, the biological sample can be processed as described above. Likewise, the at least one myoblastic differentiation biomarker can be examined at the nucleic acid or amino acid level as described above.

[00225] The methods also can include morphologically evaluating cells under a microscope {e.g., electron microscopy or light microscopy) for indicators to confirm the prognosis. Examples of indicators include, but are not limited to, cross-striations by light microscopy, actin and myosin filaments, and Z bands.

[00226] The methods also can include selecting or adjusting an appropriate therapy in an individual based upon whether there is a poor prognosis {e.g., initiating a therapy, adding a therapy, increasing a therapy and/or switching a therapy) or a good/favorable prognosis {e.g., initiating a therapy, removing a therapy, decreasing a therapy and/or switching a therapy).

[00227] Polynucleotide and polypeptide myoblastic differentiation biomarkers for use in monitoring progression of a tumor or therapeutic effectiveness can include p63, desmin, myogenin, MyoD1 and myoglobin. [00228] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin, myogenin and MyoD1 .

[00229] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin, myogenin and myoglobin.

[00230] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin, MyoD1 and myoglobin.

[00231] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, myogenin, MyoD1 and myoglobin.

[00232] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin and myogenin.

[00233] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin and MyoD1 .

[00234] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, desmin and myoglobin.

[00235] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, myogenin and MyoD1 .

[00236] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, myogenin and myoglobin.

[00237] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63, MyoD1 and myoglobin.

[00238] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 and desmin.

[00239] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 and myogenin.

[00240] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 and MyoD1 .

[00241] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 and myoglobin.

[00242] Alternatively, polynucleotide and polypeptide biomarkers for use herein can include p63 only. [00243] With respect to monitoring a tumor, preferred biomarkers include at least p63, where cytoplasmic expression of p63 indicates whether a tumor has progressed or a therapy is effective. Greater or more prevalent levels of cytoplasmic p63 expression in subsequent sample(s) generally indicate tumor progression or that therapy is ineffective, whereas attenuated or reduced levels of cytoplasmic expression of p63 in subsequent sample(s) generally indicate tumor regression or that therapy is effective. p63 preferably can be detected at the amino acid/protein level because cytoplasmic expression of p63 is being assessed. As such, one of skill in the art typically would use ELISA, IP, IF, flow cytometry or IHC to examine cytoplasmic p63 expression, preferably IHC.

[00244] Treating/Therapy Methods

[00245] The biomarkers, kits, and detecting, diagnosing, differentiating, prognosing and monitoring methods described herein can be used to assist a clinician in selecting an appropriate treatment regimen/therapy and to identify individuals that would benefit from more aggressive therapy.

[00246] As noted above, approaches to treating tumors having a skeletal muscle origin include surgery, chemotherapy, radiation therapy, a combination of chemotherapy and radiation therapy, or even an immunological or biological therapy.

EXAMPLES

[00247] The invention will be more fully understood upon consideration of the following non-limiting examples, which are offered for purposes of illustration, not limitation.

[00248] EXAMPLE 1 : RMS and Related Tumors Having a Skeletal Muscle Origin Express Cytoplasmic p63 When Compared to Other Tumors Not Having a Skeletal Muscle Origin.

[00249] Materials and Methods.

[00250] Immunohistochemistry

[00251] Thirty-eight RMS from thirty-four individuals were selected from the archives of the Department of Pathology at Indiana University School of Medicine (Indianapolis, IN). The RMS included twenty embryonal (including seven spindle cell variants), five alveolar, five pleomorphic, four mixed embryonal and alveolar, two with cytodifferentiation, and two unspecified. In addition, five leiomyomas, five leiomyosarcomas, five rhabdomyomas, five rhabdomyomatous Wilms tumors, three cases of normal cardiac muscle, one medullomyoblastoma, one pleuropulmonary blastoma with rhabdomyomatous differentiation, and one teratoma with atypical rhabdoblasts were retrieved. All cases were reviewed, along with appropriate immunostains, and the diagnoses confirmed. Formalin-fixed, paraffin -em bedded tissue blocks were cut in 4 μιτι sections and picked up onto positively charged slides. The sections then were deparaffinized and rehydrated. Antibodies against desmin (Dako, R606, pre-dilute) and p63 (Dako, M7247, 1 :60) were applied to sections from each case.

[00252] The p63 antigen was retrieved in 1 mM EDTA buffer, pH 8, by heating in a pressure cooker on high for 15 minutes. Primary antibody was diluted 1 :60 and incubated 30 minutes with the section. Detection was accomplished using the LSAB2 method (15 minutes each, Dako). Desmin antigen was retrieved in Dako's "PT Module" with its high pH Target Retrieval Solution using Dako's Flex+Mouse avidin-biotin system.

[00253] Primary antibody and all subsequent incubations were 10 minutes each. Horseradish peroxidase conjugated to the final reagent was used to develop the brown diaminobenzidine chromagen.

[00254] Immunostains were qualitatively reviewed and assessed for the presence or absence of staining. Unlike the nuclear staining scored in myoepithelial cells, only cytoplasmic staining for p63 was considered positive. p63 staining was semi- quantitatively scored for intensity on a scale of 0 to 3+, with 0 representing no cytoplasmic staining, 1 + faint cytoplasmic staining, 2+ moderate cytoplasmic staining, and 3+ intense cytoplasmic staining. Percent positive cells were not recorded. Presence or absence of visible striations was noted. Desmin was used as a comparison to p63 and also was scored on a scale of 0 to 3+, based on intensity. [00255] Immunoelectron Microscopy

[00256] Normal skeletal muscle tissue was fixed with 4% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, dehydrated through a graded series of ethyl alcohols and embedded in Unicryl (Vector Labs; Burlingame, CA). Thin sections (70-90 nm) were mounted on Formvar/carbon coated nickel grids.

[00257] After drying, the grids were floated on drops of 0.05 M glycine for 15 minutes to quench the aldehydes. After rinsing with 0.1 M phosphate buffer, the grids were placed into the blocking buffer for 30-45 minutes to block and permeabilize the tissue. The grids then were incubated in p63 antibody (1 :5 dilution) at 4°C overnight, rinsed with the incubation buffer, and floated on drops of secondary antibody labeled with 10 nm gold particles (Aurion, Electron Microscopy Sciences; Hatfield, PA) for two hours at room temperature. The grids were rinsed again in buffer and placed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for five minutes. The grids were finally rinsed in distilled water, allowed to dry, and stained for contrast with uranyl acetate. The skeletal muscle samples were viewed with a Tecnai Bio (Tecnai G2 Spirit Bio [Twin]).

[00258] Results.

[00259] Of 38 RMS, 36 (95%) showed cytoplasmic p63 staining; 24 had intense staining (3+); 9 moderate (2+); and 3 faint (1 +) (see also, Table 4 below). p63 clearly highlighted the cross-striations characteristic of skeletal muscle differentiation in 24 (63%) of the 38 cases (FIG. 1 a-b). In comparison, cytoplasmic desmin staining was seen in 35 of 38 RMS, but only 2 displayed somewhat distinct cross-striations. In addition, 5/5 rhabdomyomas (three 3+, one 2+, one 1 +), 5/5 rhabdomyomatous Wilms tumors (four 3+, one 1 +), 1/1 pleuropulmonary blastoma with rhabdomyomatous differentiation (3+), 1/1 teratoma with atypical rhabdoblasts (2+), and 1/1 medullomyoblastoma (3+) exhibited cytoplasmic p63 staining.

[00260] Several of the tumor samples included adjacent normal skeletal muscle tissues that showed intense cytoplasmic immunoreactivity with prominence of the cross- striations. Likewise, normal cardiac muscle samples (3/3, two 3+, one 2+) demonstrated positive cytoplasmic staining and distinct cross-striations (FIG. 1 c-f). Smooth muscle tumors, however, exhibited only very focal and faint cytoplasmic staining in 5/5 leiomyomas (all 1+) and 4/5 leiomyosarcomas (one 3+, three 2+, one 0).

[00261] Table 4: p63 and Desmin Expression in Tumors with Muscle Differentiation.

p63 (and Desmin) Immunostaining

0 1 + 2+ 3+ Striations present

Tumors with skeletal muscle differentiation

Rhabdomyosarcoma - total (n=38) 2(3) 3(0) 9(6) 24 (29) 24 (2)

Embryonal (n=13) 0(0) 1 (0) 6(5) 6(8) 6(0)

Spindle cell (n=7) 1 (2) 2(0) 0(0) 4(5) 3(0)

Alveolar (n=5) 0(0) 0(0) 2(0) 3(5) 3(0)

Pleomorphic (n=5) 0(0) 0(0) 1 (0) 4(5) 5(2)

Mixed embryonal and alveolar (n=4) 1 (1) 0(0) 0(0) 3(3) 3(0)

With cytodifferentiation (n=2) 0(0) 0(0) 0(0) 2(2) 2(0)

Not specified (n=2) 0(0) 0(0) 0(1) 2(1) 2(0)

Rhabdomyoma (n=5) 0(0) 1 (0) 1 (0) 3(5) 4(2)

Rhabdomyomatous Wilms (n=5) 0(0) 1 (0) 0(0) 4(5) 5(2)

Pleuropulmonary blastoma with 0(0) 0(0) 0(0) 1 (1) 1 (0) rhabdomyomatous differentiation (n=1)

Teratoma with atypical rhabdoblasts 0(0) 0(0) 1 (1) 0(0) 1 (0) (n=1)

Medullomyoblastoma (n=1) 0(0) 0(0) 0(0) 1 (1) 1 (1)

Tumors with smooth muscle differentiation

Leiomyoma (n=5) 0(0) 5(0) 0(0) 0(5) 0(0)

Leiomyosarcoma (n=5) 1 (0) 0(0) 3(0) 1 (5) 0(0)

Normal cardiac muscle differentiation 0(0) 0(0) 1 (0) 2(3) 3(3)

(n=3)

[00262] Sections where the strap cells ; could be identified on H&E preparation, demonstrated parallel desmin and p63 immunoreactivity; however, p63 highlighted the cross-striations of the strap cells, with a degree of definition not seen on the desmin immunostains (FIG. 2). Despite the fact that desmin was a poor marker for cross- striations, it showed diffuse and usually intense immunoreactivity in most cases (see, Table 4 above).

[00263] Skeletal muscle sections displayed p63 localization primarily to the Z lines of the sarcomeres (FIG.3). [00264] Desmin stained both skeletal and smooth muscle, and therefore cannot be used to distinguish the two. p63, however, highlights cross-striations not seen with desmin immunohistochemistry (FIG. 4).

[00265] p63 immunostain therefore is a sensitive myoblastic differentiation biomarker. It highlights the cross-striations of strap cells with exceptional definition, much superior to desmin. The high frequency of distinct cross-striations on immunohistochemistry, in conjunction with the immunoelectron microscopy study supports that p63 localizes to the Z lines of skeletal and cardiac muscle. Desmin has been reported to be concentrated in the bundles of intermediate filaments connecting Z lines to adjacent myofibrils. In addition to being present between myofibrils, desmin filaments also are found within myofibrils, which can explain its more diffuse staining. Smooth muscle cells also contain desmin intermediate filaments and stain with desmin immunohistochemistry.

[00266] All of the patents, patent applications, patent application publications and other publications recited herein are hereby incorporated by reference as if set forth in their entirety.

[00267] The present invention has been described in connection with what are presently considered to be the most practical and preferred embodiments. However, the invention has been presented by way of illustration and is not intended to be limited to the disclosed embodiments. Accordingly, one of skill in the art will realize that the invention is intended to encompass all modifications and alternative arrangements within the spirit and scope of the invention as set forth in the appended claims.

Claims

CLAIMS The invention claimed is:

1 . A method of monitoring progression of a tumor having a skeletal muscle origin in an individual having the tumor, the method comprising the step of:

detecting cytoplasmic expression of at least one myoblastic differentiation biomarker in cells from first and second chronological biological samples from the individual, wherein elevated cytoplasmic expression of the at least one biomarker in cells of the second chronological sample when compared to cytoplasmic expression of the at least biomarker in cells of the first chronological sample indicates tumor progression, or

wherein decreased cytoplasmic expression of the at least one biomarker in cells of the second chronological sample when compared to cytoplasmic expression of the at least biomarker in cells of the first chronological sample indicates tumor regression.

2. A method of monitoring efficacy of a therapy in an individual having a tumor having a skeletal muscle origin, the method comprising the step of:

determining cytoplasmic expression of at least one myoblastic differentiation biomarker in cells from at least two chronological biological samples from the individual, wherein elevated cytoplasmic expression of the at least one biomarker in cells in a later- obtained chronological sample when compared to cytoplasmic expression of the at least biomarker in cells in an earlier-obtained chronological sample indicates an ineffectiveness of therapy, or

wherein attenuated cytoplasmic expression of the at least one biomarker in cells in a later-obtained chronological sample when compared to cytoplasmic expression of the at least biomarker in cells in an earlier-obtained chronological sample indicates effectiveness of therapy.

3. The method of Claim 1 or 2, further comprising the step of obtaining the biological sample from the individual.

4. The method of any of Claims 1 -3, further comprising the step of processing the biological sample.

5. The method of any of Claims 1 -4, further comprising the step of morphologically evaluating cells under a microscope to confirm progression of the tumor or effectiveness of the therapy.

6. The method of Claims 1 -5, further comprising the step of selecting an additional therapy or adjusting the therapy in the individual based upon effectiveness of the therapy.

7. The method of any of Claims 1 -6, wherein the at least one myoblastic differentiation biomarker is selected from the group consisting of p63, desmin, myogenin, MyoD1 and myoglobin.

8. The method of any of Claims 1 -6, wherein the at least one myoblastic differentiation biomarker is p63 and an additional biomarker selected from the group consisting of desmin, myogenin, MyoD1 and myoglobin.

9. The method of any of Claims 1 -6, wherein the at least one myoblastic differentiation biomarker is p63.

10. The method of any of Claims 1 -9, wherein the tumor having the skeletal muscle origin is selected from the group consisting of rhabdomyosarcoma, rhabdomyomas, rhabdomyomatous Wilms tumors, pleuropulmonary blastoma with rhabdomyomatous differentiation, teratoma with atypical rhabdoblasts, medullomyoblastoma and Triton tumor (peripheral nerve sheath tumor with striated muscle differentiation).

1 1 . A method of prognosing an individual having a tumor having a skeletal muscle origin, the method comprising the step of:

detecting cytoplasmic expression of at least one myoblastic biomarker in cells from the individual, wherein elevated cytoplasmic expression of the biomarker when compared to a control or reference indicates a poor prognosis, or

wherein attenuated cytoplasmic expression of the biomarker when compared to a control or reference indicates a good/favorable prognosis.

12. The method of Claim 1 1 , further comprising the step of obtaining the biological sample from the individual.

13. The method of Claims 1 1 or 12, further comprising the step of processing the biological sample.

14. The method of any of Claims 1 1 -13, further comprising the step of morphologically evaluating cells under a microscope to confirm prognosis.

15. The method of any of Claims 1 1 -14, further comprising the step of selecting or adjusting a therapy in the individual based upon prognosis.

16. The method of any of Claims 1 1 -15, wherein the at least one myoblastic differentiation biomarker is selected from the group consisting of p63, desmin, myogenin, MyoD1 and myoglobin.

17. The method of any of Claims 11 -15, wherein the at least one myoblastic differentiation biomarker is p63 and an additional biomarker selected from the group consisting of desmin, myogenin, MyoD1 and myoglobin.

18. The method of any of Claims 11 -15, wherein the at least one myoblastic differentiation biomarker is p63.

19. The method of any of Claims 1 1 -18, wherein the tumor having the skeletal muscle origin is selected from the group consisting of rhabdomyosarcoma, rhabdomyomas, rhabdomyomatous Wilms tumors, pleuropulmonary blastoma with rhabdomyomatous differentiation, teratoma with atypical rhabdoblasts, medullomyoblastoma and Triton tumor (peripheral nerve sheath tumor with striated muscle differentiation).

20. A method of differentiating tumors having a skeletal muscle origin from tumors that do not have a skeletal muscle origin, the method comprising the step of:

detecting cytoplasmic expression of at least one myoblastic differentiation biomarker in cells from a biological sample suspected of having a tumor having a skeletal muscle origin, wherein cytoplasmic expression of the at least one biomarker indicates that the tumor has a skeletal muscle origin, or

wherein lack of cytoplasmic expression of the at least one biomarker indicates that the tumor does not have a skeletal muscle origin.

21 . The method of Claim 20, further comprising the step of obtaining the biological sample from the individual.

22. The method of Claims 20 or 21 , further comprising the step of processing the biological sample.

23. The method of any of Claims 20-22, wherein the at least one myoblastic differentiation biomarker is selected from the group consisting of p63, desmin, myogenin, MyoD1 and myoglobin.

24. The method of any of Claims 20-22, wherein the at least one myoblastic differentiation biomarker is p63 and an additional biomarker selected from the group consisting of desmin, myogenin, MyoD1 and myoglobin.

25. The method of any of Claims 20-22, wherein the at least one myoblastic differentiation biomarker is p63.

26. The method of any of Claims 20-25, wherein the tumor having the skeletal muscle origin is selected from the group consisting of rhabdomyosarcoma, rhabdomyomas, rhabdomyomatous Wilms tumors, pleuropulmonary blastoma with rhabdomyomatous differentiation, teratoma with atypical rhabdoblasts, medullomyoblastoma and Triton tumor (peripheral nerve sheath tumor with striated muscle differentiation).

27. A method of diagnosing an individual as having a tumor having a skeletal muscle origin, the method comprising the step of:

detecting cytoplasmic expression of at least one myoblastic differentiation biomarker in cells from a biological sample from the individual, wherein cytoplasmic expression of the at least one biomarker in the cells indicates that individual has the tumor having a skeletal muscle origin.

28. The method of Claim 27, further comprising the step of obtaining the biological sample from the individual.

29. The method of Claims 27 or 28, further comprising the step of processing the biological sample.

30. The method of any of Claims 27-29, wherein the at least one myoblastic differentiation biomarker is selected from the group consisting of p63, desmin, myogenin, MyoD1 and myoglobin.

31 . The method of any of Claims 27-29, wherein the at least one myoblastic differentiation biomarker is p63 and an additional biomarker selected from the group consisting of desmin, myogenin, MyoD1 and myoglobin.

32. The method of any of Claims 27-29, wherein the at least one myoblastic differentiation biomarker is p63.

33. The method of any of Claims 27-32, wherein the tumor having the skeletal muscle origin is selected from the group consisting of rhabdomyosarcoma, rhabdomyomas, rhabdomyomatous Wilms tumors, pleuropulmonary blastoma with rhabdomyomatous differentiation, teratoma with atypical rhabdoblasts, medullomyoblastoma and Triton tumor (peripheral nerve sheath tumor with striated muscle differentiation).

34. A method of detecting myoblastic differentiation in cells suspected of undergoing myoblastic differentiation, the method comprising the step of:

detecting cytoplasmic expression of at least one myoblastic differentiation biomarker in cells, wherein cytoplasmic expression of the at least one biomarker in the cells indicates that the cells are undergoing myoblastic differentiation.

35. The method of Claim 34, further comprising the step of obtaining the biological sample from an individual having or suspected of having cells undergoing myoblastic differentiation.

36. The method of Claims 34 or 35, further comprising the step of processing the biological sample.

37. The method of any of Claims 34-36, wherein the at least one myoblastic differentiation biomarker is selected from the group consisting of p63, desmin, myogenin, MyoD1 and myoglobin.

38. The method of any of Claims 34-36, wherein the at least one myoblastic differentiation biomarker is p63 and an additional biomarker selected from the group consisting of desmin, myogenin, MyoD1 and myoglobin.

39. The method of any of Claims 34-36, wherein the at least one myoblastic differentiation biomarker is p63.

40. The method of any of Claims 34-39, wherein the tumor having the skeletal muscle origin is selected from the group consisting of rhabdomyosarcoma, rhabdomyomas, rhabdomyomatous Wilms tumors, pleuropulmonary blastoma with rhabdomyomatous differentiation, teratoma with atypical rhabdoblasts, medullomyoblastoma and Triton tumor (peripheral nerve sheath tumor with striated muscle differentiation).

41 . The method of any of Claims 1 -40, wherein cytoplasmic expression of the at least one myoblastic differentiation biomarker is detected with a nucleic acid probe that hybridizes to a nucleic acid molecule that expresses the biomarker.

42. The method of any of Claims 1 -40, wherein cytoplasmic expression of the at least myoblastic differentiation biomarker is detected with an antibody that binds to the biomarker.

43. Use of p63 as a cytoplasmic biomarker for monitoring effectiveness of a therapy in an individual having a tumor having a skeletal muscle origin.

44. Use of p63 as a cytoplasmic biomarker for prognosing an individual having a tumor having a skeletal muscle origin.

45. Use of p63 as a cytoplasmic biomarker for differentiating tumors having a skeletal muscle origin from tumors that do not have a skeletal muscle origin.

46. Use of p63 as a cytoplasmic biomarker for diagnosing an individuals has having a tumor having a skeletal muscle origin.

47. Use of p63 as a cytoplasmic biomarker for detecting myoblastic differentiation in cells suspected of undergoing myoblastic differentiation.

48. Use of any of Claims 43-47, further comprising use of at least one of desmin, myogenin, MyoDI and myoglobin for detecting myoblastic differentiation.

49. A kit for evaluating expression myoblastic differentiation, the kit comprising:

means for detecting cytoplasmic expression of a p63 myoblastic differentiation biomarker and at least one additional myoblastic differentiation biomarker; and

a positive control and optionally a negative control, wherein the positive control has cytoplasmic expression of p63, and wherein the optional negative control, if included, does not have cytoplasmic expression of p63 or does not express p63.

50. The kit of Claim 49, wherein the means for detecting cytoplasmic expression is a nucleic acid probe that hybridizes to a nucleic acid molecule that expresses p63 or the at least one additional biomarker.

51 . The kit of Claim 49, wherein the means for detecting cytoplasmic expression is an antibody that binds to p63 or the at least one additional biomarker.

52. The kit of Claim 49, further comprising a means for processing a biological sample.

53. The kit of Claim 52, wherein the means for processing a biological sample is a buffer selected from the group consisting of a cell isolation buffer, fixation buffer, lysis buffer, permeabilization buffer, sonication buffer, separation buffer, stabilization buffer and wash buffer.

54. The kit of any of Claims 49-51 , wherein the at least one additional myoblastic differentiation biomarker is selected from the group consisting of myogenin, MyoDI, desmin and myoglobin.